TECHNICAL FIELD
This invention relates to a TRAF family molecule,
a polynucleotide encoding the molecule, an antibody
against the molecule, and an antisense polynucleotide
of the polynucleotide encoding the molecule.
BACKGROUND ART
For molecules belonging to the TNF receptor
family (hereinafter referred to as "TNF-R"), TNF-R1,
TNF-R2, Fas, CD40, CD27, and lymphotoxin-β receptors
(hereinafter referred to as "LT-βR" or "TNF-Rrp") are
known. These molecules are provided with the function
of finally causing the phenomena of the proliferation,
cell differentiation, and necrosis of cells by binding
to their ligands, or with the function as second
signal molecules of other molecules.
Also, TRAF1 and TRAF2 (the abbreviation of TNF-R
associated factor), which are molecules capable of
associating TNF-R, have been cloned (Goeddel et. al,
Cell Vol. 78, No. 4, p. 681-692, 1994) and these are
known to associate the domain within the cytoplasm of
TNF-R2.
Furthermore, two additional kinds of new TRAF
molecules have been identified and the following are
noted for these molecules:
(1) TRAF1 associates TNF-R2 and CD40. (J. Biol.
Chem. Vol. 269, No. 48, p. 30069-30072.) (2) TRAF2 plays an important role in activation
of the DNA-binding of NF-κB by TNF-R2 or CD40: NF-κB
is a nucleoprotein binding to a base sequence
comprising 10 base pairs that is within the enhancer
region of the Igκ L chain gene and is referred to as
"κB motif". (3) TRAF3 (also referred to as "CRAF1," "CD40bp,"
or "CAP1") is known to associate TNF-R2, CD40, and LT-βR.
(Science Vol. 267, No. 5203, p. 1494-1498; FEBS
Lett. Vol. 358, No. 2, p. 113-118; J. Biol. Chem. Vol.
268, p.30069-30072; and Cell Vol. 80, No. 3, P. 389-399.) (4) As for TRAF4 (also referred to as "CART1"),
no molecule has yet been identified to associate it.
(J. Biol. Chem. Vol. 270, No. 4, P. 25715-25721,
1995.)
Moreover, it has been reported that neither
TRAF1 nor TRAF3 induces activation of the DNA-binding
of NF-κB. (Cell Vol. 81, p.495-504; Science Vol. 269,
p.1424-1427; and Proc. Natl. Acad. Sci. USA, Vol. 93,
p.9699-9703.)
Molecules of the TRAF family are structurally
characterized by having TRAF domains, and some members
of the family molecules have ring finger motifs, Zn
finger motifs or leucine zipper motifs at their N-termini,
or coiled coil structures at their centers.
Because of these structural features, it is presumed
that these TRAF family molecules form complexes in a
homo- or hetero-fashion and function as transcription
factors.
DISCLOSURE OF THE INVENTION
An object of this invention is to provide a novel
TRAF family molecule. Another object of the invention
is to provide a substance capable of having an
application in the elucidation of functions of the
TRAF family molecules, such as an antibody or a
polynucleotide probe: namely, the elucidation of
interaction between the TRAF family molecules and the
elucidation of the signal transduction system of the
TNF-R family. A further object of the invention is to
provide an antisense polynucleotide applicable to the
development of pharmaceuticals.
Specifically, an object of the invention is to
provide the screening for the TRAF5 molecule, which is
a novel TRAF family molecule according to the
invention, isolation and structural determination
thereof, as well as to provide a variety of functions
that the molecule possesses.
In other words, the object of the invention is as
follows: homology with respect to TRAF1 and TRAF2 is
analyzed by computer; various oligo-DNA primers
capable of amplifying the region that is most highly
conserved are synthesized; cDNA is prepared from RNA
that is derived from different kinds of cell strains
and tissues; PCR is performed using the oligo-DNA
primers and the cDNA as a template; and the thus
obtained novel gene (TRAF5 gene) exhibiting a high
degree of homology to the TRAF1 and TRAF2 genes as
well as the amino acid sequence of the TRAF5 molecule
encoded by the gene is provided.
In the present specification, the protein will be
hereinafter referred to as "TRAF5 molecule" or "TRAF5"
and the TRAF5 gene itself referred to as "TRAF5 gene."
It is also an object of the invention to provide
information that the TRAF5 molecule tends to associate
LT-βR and CD30 and further that it has the ability to
induce the DNA-binding activity of NF-κB, which is a
DNA-binding protein.
In addition, an object of the invention is to
provide means for elucidating the functions of the
TRAF5 molecule as well as to provide means for
elucidating the signal transduction system of the TNF-R
family involving the TRAF family molecules by
preparing antibodies against the TRAF5 molecule and
using the antibodies.
A still further object of the invention is to
provide means for making the TRAF5 gene applicable to
experimental probes, probes diagnosing the gene or the
antisense genes, and therapeutic agents.
More specifically, this invention provides novel
TRAF family molecules as described below, including
the TRAF5 molecule.
1. A TRAF family molecule having the properties
as noted in (1)-(4) in the following:
(1) Having the ability to associate lymphotoxin-β
receptors (LT-βR) and CD30; (2) Having the ability to induce the DNA-binding
activity of a DNA-binding protein; (3) Having the inability to associate CD40 or
TNF-R2; and (4) Having a leucine zipper motif or coiled coil
structure within its molecule. 2. The TRAF family molecule as described above
wherein the DNA-binding protein is NF-κB. 3. The TRAF family molecule as described above
wherein said molecule is a protein encoded by DNA
hybridizing to the base sequence set forth in SEQ ID
No. 2 of the Sequence Listing under stringent
conditions. 4. The TRAF family molecule as described above
wherein said molecule has at least part of the amino
acid sequence set forth in SEQ ID No. 1 or SEQ ID No.
3 of the Sequence Listing. 5. A TRAF5 molecule comprising the amino acid
sequence set forth in SEQ ID No. 1 of the Sequence
Listing. 6. A TRAF5 molecule comprising the amino acid
sequence set forth in SEQ ID No. 3 of the Sequence
Listing. 7. A TRAF family molecule comprising a modified
TRAF5 molecule, said TRAF5 molecule comprising the
amino acid sequence which is derived by modifying a
part of the amino acid sequence set forth in SEQ ID No.
1 or SEQ ID No. 3 of the Sequence Listing, by
substitution, deletion or addition of amino acid(s)
without substantially altering the functions of the
TRAF family molecule. 8. A TRAF5 molecule comprising an amino acid
sequence of from position 233 to position 558 of the
amino acid sequence set forth in SEQ ID No. 1 of the
Sequence Listing. 9. A TRAF5 molecule comprising an amino acid
sequence of from position 342 to position 558 of the
amino acid sequence set forth in SEQ ID No. 1 of the
Sequence Listing. 10. A TRAF5 molecule comprising an amino acid
sequence of from position 233 to position 557 of the
amino acid sequence set forth in SEQ ID No. 3 of the
Sequence Listing. 11. A TRAF5 molecule comprising an amino acid
sequence of from position 342 to position 557 of the
amino acid sequence set forth in SEQ ID No. 3 of the
Sequence Listing. 12. A polynucleotide encoding the TRAF family
molecule as described above. 13. A TRAF5 gene comprising the base sequence set
forth in SEQ ID No. 2 of the Sequence Listing. 14. A TRAF5 gene comprising the base sequence set
forth in SEQ ID No. 4 of the Sequence Listing. 15. A polynucleotide comprising part of the
polynucleotide according to any of Items 12-14 as
described above, said part being 12 or more
consecutive bases. 16. A polynucleotide comprising part of the
polynucleotide according to any of Items 12-14 as
described above, said part being 16 or more
consecutive bases. 17. An antibody directed against the TRAF family
molecule according to any of Items 1-11 as described
above. 18. An expression vector comprising a
polynucleotide encoding the TRAF family molecule
according to Item 12 as described above. 19. A transformant characterized by being
transformed with the expression vector as described
above. 20. A method of producing a TRAF family molecule,
said method comprising using the transformant as
described above. 21. An antisense polynucleotide of a
polynucleotide encoding the TRAF family molecule
according to Item 12 as described above. 22. A polynucleotide comprising part of the
antisense polynucleotide as described above, said part
being 12 or more consecutive bases. 23. A polynucleotide comprising part of the
antisense polynucleotide as described above, said part
being from 16 or more to 30 or less consecutive bases. 24. A derivative of the polynucleotide according
to any of Items 12-16 or Items 21-23 as described
above.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing the results of a
homology analysis of the amino acid sequences for the
TRAF5 molecule and other TRAF family molecules, where
the blacked part represents a TRAF domain, the
vertically lined part represents a leucine zipper
motif, the obliquely lined part represents a Zn finger
motif, and the horizontally lined part represents a Zn
ring finger motif; with respect to TRAF1-TRAF4, amino
acid homology in their overall sequences and their
TRAF domains against TRAF5 is indicated on the right-hand
side.
FIG. 2 is a photograph showing the results of a
northern blot hybridization analysis of the murine
TRAF5 gene in respective tissues, where the arrow
indicates the position of a band of the murine TRAF5
gene.
FIG. 3 is a photograph showing the results of
SDS-PAGE from screening for any TNF-R family molecules
that associate the murine TRAF5 molecule, where the
arrow indicates the position of a band of the murine
TRAF5 molecule.
FIG. 4 is a photograph showing the results of
immunoprecipitation western blotting by means of a HA
epitope, of the associated substance between the
murine TRAF5 molecule and LT-βR, where the arrow
indicates the position of the murine TRAF5 molecule
bound to the HA epitope.
FIG. 5 is a photograph showing the results of an
EMSA that illustrates induction of the DNA-binding
activity of NF-κB by the murine TRAF5 molecule or the
LT-βR molecule, where the band as indicated by "B→"
indicates the position of NF-κB and the depth of the
band in color indicates the quantity of DNA bound to
NF-κB.
FIG. 6A is a graph showing a primer used to
identify the murine TRAF5 gene.
FIG. 6B is a graph showing a primer used to
identify the murine TRAF5 gene.
FIG. 7 is a graph showing the results of an ELISA
of the polyclonal antibody against the 14mer peptide
(sequence 1 peptide) having Cys (C) appended to the N-terminus
of the TRAF5 molecule: Ser Val Lsy Gln Arg
Ile Thr Gln Leu Glu Ala Ser Asp (SVKQRITQLEASD), i.e.,
positions 351-363.
FIG. 8 is a graph showing the results of an ELISA
of the polyclonal antibody against the 14mer peptide
(Sequence 2 peptide) having Cys Tyr Ser Gly Lys Leu
Ile Trp Lys Val Thr Asp Tyr Arg (CYSGKLIWKVTDYR):
positions 401-414 of the murine TRAF5 molecule.
FIG. 9 is a photograph of western blotting
showing expression of the human TRAF5 molecule in
respective cell strains, where the arrow indicates the
position of a band of the human TRAF5 molecule.
FIG. 10 is a photograph showing that CD30 or LT-β
R associates the murine TRAF5 molecule or the murine
TRAF2 molecule or the murine TRAF3 molecule, where the
arrow indicates the position of the band of the murine
TRAF2 molecule, the murine TRAF3 molecule or the
murine TRAF5 molecule.
FIG. 11 is a graph Showing a comparison between
the amino acid sequence of the human TRAF5 molecule
and the amino acid sequence of the murine TRAF5
molecule, where the upper row represents the amino
acid of the human TRAF5 molecule and the lower row
represents the amino acid of the murine TRAF5
molecule; "-" in the murine TRAF sequence indicates
the amino acid at an indicated position being
identical with its corresponding amino acid of human
TRAF5 (i.e., the amino acid depicted in the upper
row); and "-" at number 494 of the human TRAF5
sequence indicates deletion of an amino acid at the
position in question.
BEST MODE FOR CARRYING OUT THE INVENTION
This invention will be explained in detail
hereinbelow.
(Identification of Novel TRAF Family Molecules)
The novel TRAF family molecules according to this
invention and methods for identifying polynucleotides
encoding the TRAF family molecules according to the
invention will concretely be illustrated below.
First, in order to find a high homology region in
TRAF family molecules, the known base sequences of
TRAF1 and TRAF2 are screened for homology and the high
homology region between the two is specified for
selection.
Secondly, primers are designed to perform the PCR
amplification (polymerase chain reaction) of the
specified region. The base sequences of the primers
for such a purpose are not particularly limited, but
for example, preferably usable is a mixture of primers
of 17mer bases to which EcoRI sites are appended, as
shown in FIGs. 6A and 6B. In addition, such primers
can be synthesized using a standard DNA synthesizer.
In FIG. 6A "A" represents adenine, "T" thymine,
"G" guanine, "C" cytosine, "Y" cytosine or thymine,
"N" adenine or thymine or guanine or cytosine, "R"
adenine or guanine, and "D" any base of adenine,
thymine and guanine. Thus, (1) in FIG. 6A represents
256 kinds of primers and (2) in FIG. 6B represents 768
kinds of primers.
Furthermore, the selection of DNA that serves as
a template is not particularly limited in this
invention, but in practice mRNA may preferably be
prepared from murine B cell lymphoma, murine monocyte
cell strains, and murine liver, and then cDNA may
preferably be prepared using oligo-dT or a random
hexamer as a primer.
In addition, the cells for the purpose of
preparation of the mRNA are not limited to the above-mentioned
cells in the invention.
Preparation of the mRNA, for example, can
preferably be carried out with ease using an Oligo-dT
cellulose column after total RNA fractions have been
obtained by the thiocyanic acid guanidine method or
the like (Biochemistry Vol. 13, p. 2633, 1974).
The PCR is performed using the above-mentioned
primers and the cDNA pool obtained as described above
as a template. The amplified fragment is then
recovered and is subcloned into a cloning vector
having a muticloning site: pBluescript SK(+) is
preferably usable, for example. DNA sequencing is
conducted on this subcloned product with a DNA
sequencer according to the dideoxy method, revealing
it to be a novel gene. Such method will provide novel
TRAF family gene fragments. This novel TRAF family
gene is labeled with 32P to give one that serves as 32P-TRAF
fragment. In carrying out the dideoxy method,
either a primer method relying on primers labeled with
fluorescent dyes or a dye terminator method relying on
ddNTP labeled with fluorescent dyes can preferably be
used.
On the other hand, Poly(A)RNA is newly prepared
from a monocyte cell strain or a tissue such as liver
tissue and then, a cDNA library is constructed from
this. Any of phage vectors such as λgt10, λgt11, and
λZAP11 and plasmid vectors such as pBR and pUC is
usable for this cDNA library.
The cDNA library thus prepared is screened using
the aforementioned 32P-TRAF fragment as a probe, and a
clone containing cDNA that covers the whole
translational region of TRAF5 can be obtained. Next,
its base sequence is determined using a DNA sequencer.
Further, specific examples for the method to
obtain a murine TRAF5 gene are shown in the Examples
of the present specification as will be illustrated
below. It is possible to screen for and obtain TRAF5
genes from other animals, including human, by
employing cDNA libraries that are prepared from the
cells of desired animal species in a similar manner to
the method as described above.
The structure of a TRAF5 gene, for example in the
murine case, is shown as SEQ ID No. 2 of the Sequence
Listing. The murine TRAF5 comprises an open reading
frame of the 1674 residue nucleotide (a portion from
position 323 to position 1999 of the SEQ ID of the
Sequence Listing), namely 558 amino acid residues.
The size of its mRNA is assumed to be 2.2 kb.
The amino acid sequence of the murine TRAF5
molecule encoded by the above-mentioned base sequence
is shown in SEQ ID No. 1 of the Sequence Listing.
Referring to FIG. 1, the amino acid sequence is
compared to the known TRAF family molecules.
Structurally, the TRAF5 molecule resembles the TRAF3
molecule. The TRAF5 molecule differs from the TRAF1,
TRAF2, or TRAF4 molecule in that it has a leucine
zipper motif or a coiled coil structure.
Also, as FIG. 11 shows, very high homology exists
between the amino acid sequence of the murine TRAF5
molecule and the amino acid sequence of the human
TRAF5 molecule and its functions are well preserved
between the different species.
The TRAF5 genes show high homology between a
mouse and human. Therefore, a TRAF5 gene can also be
obtained from the desired animal species, according to
the method of Example 7 as will be described later:
namely, the method in which part or the whole of the
TRAF5 gene already in possession is used as a probe to
isolate the TRAF5 gene of the desired animal from a
cDNA library that originates in the animal by
following a cDNA cloning method such as the plaque
hybridization method, which is conventionally employed.
(Analysis of the Functions of TRAF Family Molecules of This Invention)
Screening is possible for TNF-R family molecules
that the TRAF5 molecules of the invention tend to
associate, according to the method as described below,
for example.
First, a fusion protein is prepared using an
expression vector system for the fusion protein.
Specifically, preferably usable is a method in which
the domain part within the cytoplasm of a TNF-R family
molecule is inserted into the downstream region of
glutathione-S-transferase (GST) to prepare the fusion
protein. In this invention, those other than GST
which can synthesize fusion proteins with TNF-R family
molecules are usable. Concretely, expression systems
for thioredoxin fusion proteins, e.g., Thio Fusion
(Registered Trademark, available from Invitrogen Inc.)
and for fusion proteins with leader peptides derivable
from gene 10 of T7 can also be used.
Moreover, to easily detect the TRAF5 molecule,
the TRAF5 gene is translated in vitro to prepare a
TRAF5 molecule labeled with 35S-methionine or the like,
which enables detection of the TRAF5 molecule.
Next, fusion proteins and the TRAF5 molecule are
mixed and a fusion protein having associated the TRAF5
molecule is selected. For the method of selection, a
method to have any associated substance adsorbed onto
a GSH-agarose column can be used, for example, and in
addition, chromatographic operations such as affinity
chromatography can also preferably be used.
To ascertain the ability to associate, after the
associated substance has been separated and heated to
dissociate the TRAF5 molecule, SDS-PAGE is conducted
and the gel is autoradiographed, thus detecting the
presence of the band of the TRAF5 molecule. Thereby,
it becomes possible to ascertain that the TRAF5
molecule has the ability to associate LT-βR and CD30.
Also, after both the TRAF5 molecule and LT-βR
have been forced to express in the same animal cells,
any associated substance in the cells is subjected to
the immunoprecipitation blotting: this method also
enables confirmation of the association between the
TRAF5 molecule and LT-βR. Specifically for this
purpose, the TRAF5 molecule to be used should
preferably be bound to the hemagglutinin epitope (HA
epitope) of influenza virus which serves as a tag.
Alternatively, preferably usable is a method in which
the leader peptide derivable from gene 10 of T7 as
described previously has been bound to the molecule.
Further, although a variety of methods known in the
art can be used for the method of binding a tag as
described previously, genetic engineering methods are
preferable in this invention. Especially, after
treatment with restriction enzymes such as EcoRI, DNA
encoding the HA epitope and the TRAF5 molecule that
has been amplified by PCR are preferably incorporated
into an expression vector.
Where the TRAF5 molecule is to be prepared by a
genetic engineering method, it is also preferable that
an expression vector for the TRAF5 molecule and a
vector into which LT-βR has been incorporated be
introduced into cells. As to the method for
introducing vectors into cells, a liposome method, an
electroporation method, and others can be used, but
particularly, it is preferable to use a DEAE-dextran
method (available from Pharmacia Inc.).
Furthermore, if an antibody against the epitope
bound as a tag (anti-HA antibody in the case where the
HA epitope is bound) is added to the cell product and
is allowed to react thereto, it is possible to
separately and precisely collect the TRAF5 molecule
from any substances associating the TRAF5 molecule.
As previously noted, the TRAF5 molecules of this
invention have the ability to associate LT-βR and CD30,
which are TNF-R family molecules. Such a property can
be used to elucidate the functions of TNF-R family
molecules. The TNF-R and TNF family molecules are a
group of important molecules that are the trigger for
causing the differentiation, proliferation, and death
of cells; therefore, use of the TRAF5 molecules, genes
thereof, and antibodies against them makes it possible
to elucidate such functions and then to elucidate the
mechanisms of cancer and apoptosis.
Similar to LT-α, LT-βR is also presumed to have
an important role in differentiation of the immune
system. Hitherto, it has been known that the
differentiation of peripheral lymph nodes is immature
in mice deficient in the LT-α gene, and it is believed
that so is the case with LT-βR. As for the functions
of CD30, it is known that in the deficient mice, the T
cells can not normally differentiate within their
thymi and the signals often do not transmit well
through T cell receptors. Thus, that the TRAF
molecule is a substance to be the key factor in the
signal transduction of LT-βR suggests association
between the TRAF5 molecule and LT-βR, or CD30. It is
then presumed that the mice deficient in the TRAF5
molecule cause immatureness of their immune systems,
e.g., immatureness in the differentiation of their
peripheral lymph nodes.
Next, the TRAF5 molecules of this invention are
used to examine cells' action of signal transduction.
This makes it possible to ascertain that the
expression of the TRAF5 molecule induces activation of
the DNA-binding of NF-κB which is a DNA-binding
protein. In other words, it is ascertained that the
TRAF5 molecules can be used to induce activation of
the DNA-binding of a DNA-binding protein.
In this invention, it can be also ascertained
that with the use of the TRAF5 molecule according to
the invention, the expression of LT-βR alone causes
the expression of NF-κB, but that this activation is
inhibited by adding an excess of only a portion of
from position 233 to position 558 of the murine TRAF5
molecule which is a deleted protein. Namely, by
utilizing the portion of positions 233-558 of the
TRAF5 molecule, it becomes possible to study its
action to antagonize LT-βR. The preparation of the
TRAF5 molecules or TRAF5 genes according to this
invention also enables the study on a system
controlling the signal transduction involving NF-κB,
as well as the development of drugs.
Employing a polynucleotide probe containing the
binding site of NF-κB, the NF-κB bound to the probe
is to be detected in the cells where the TRAF molecule
has been expressed: for example, this method can be
used to ascertain this. The TRAF5 molecule to be used
may be part thereof or the whole molecule. The
polynucleotide probe containing the binding site of
NF-κB may be the gene of NF-κB itself or part thereof,
and may be of natural or synthetic origin. For the
method of detection, Electrophoretic Mobility Shift
Assay (EMSA) may be employed, for example. Other than
this, the gel shift method may be usable.
As noted above, the TRAF family molecules of this
invention are provided with the property of
associating LT-βR and CD30, and capable of inducing
the DNA-binding activity of a DNA-binding protein.
For the DNA-binding protein to ascertain its DNA-binding
activity, NF-κB is preferable.
Further, among the known TRAF family molecules,
the TRAF3 molecule is structurally similar to the
TRAF5 molecule. Based on the information obtained in
this invention concerning the association with LT-βR
and CD30 in addition to the activation of NF-κB, it
has been verified that the TRAF3 molecule neither
associate TNF-R2 nor CD40 and is able to activate NF-κ
B. Thus, it is different from the TRAF5 molecules
according to this invention.
(TRAF Family Molecules of This Invention)
The properties that characterize the TRAF family
molecules of this invention are summarized in the
following four points:
(1) They have the ability to associate
lymphotoxin-βreceptors (LT-βR) and CD30. (2) They have the ability to induce the DNA-binding
activity of DNA-binding proteins. (3) They have the inability to associate CD40 or
TNF-R2. (4) They have a leucine zipper motif or a coiled
coil structure within their molecules.
Insofar as the TRAF family molecules of the
invention have the above-mentioned properties, they
embrace ones that contain at least parts of the amino
acid sequence set forth in SEQ ID No. 1 or SEQ ID No.
3 of the Sequence Listing. Although the part of the
amino acid sequence as described above is not
particularly limited, the one of from position 233 to
position 558 of the amino acid sequence set forth in
SEQ ID No. 1 of the Sequence Listing is preferable.
Especially, it may preferably have the TRAF domain
(positions 342-558 of the amino acid sequence set
forth in SEQ ID No. 1 of the Sequence Listing).
Insofar as the TRAF family molecules of the
invention have the above-mentioned properties, they
also embrace proteins encoded by DNAs hybridizing to
part of the amino acid sequence set forth in SEQ ID No.
2 of the Sequence Listing, as will be illustrated in
Example 7.
Insofar as the TRAF family molecules of the
invention have the above-mentioned properties, they
also embrace the TRAF family molecule part of which
has been structurally modified by substitution,
deletion or addition of amino acid(s). For the method
of substitution, deletion or addition of amino acid(s),
techniques known in the art are usable, for example,
as described in "Molecular Cloning 2nd edition," p.
15.1-15.113.
(The Polynucleotides of This Invention Encoding TRAF
Family Molecules)
The polynucleotides of the invention encoding
TRAF family molecule are, for example, a DNA and a RNA
corresponding to the DNA, which DNA comprises the base
sequence of from position 323 to position 1999 of the
base sequence of SEQ ID No. 2 of the Sequence Listing.
These are polynucleotides encoding the murine TRAF5
molecule comprising the amino acid sequence of SEQ ID
No. 1 of the Sequence Listing. The whole or part of
the coding region of the protein can be expressed in
suitable transformants, which can be used to produce
recombinant TRAF family molecules.
In addition, part of the polynucleotide can
immediately be used as polynucleotide probes for
research purposes that aim at investigating the
presence of genes encoding the TRAF family molecules
in tissues or cells, as well as their expression state.
Preferably, the part to be used as the probe is 12
bases or more and has a GC content of from 30 to 70%.
Most preferably, it is 16 bases or more. The
polynucleotide to be used as the probe may be a
derivative. The polynucleotide may be a base sequence
of the noncoding region of the gene coding the TRAF
family molecule or its complementary base sequence
when it is to be used as the probe.
(The Antibodies of This Invention against TRAF Family
Molecules)
The antibodies of the invention against TRAF
family molecules embrace either of polyclonal
antibodies and monoclonal antibodies insofar as they
recognize the TRAF family molecules. They also
embrace active fragments thereof and chimera
antibodies containing the active fragments.
The antibody, i.e., immunoglobulin, has H and L
chains, and is classified into five classes (IgA, IgD,
IgE, IgG, and IgM) based on its physicochemical
immunological properties. Among these, IgA and IgG
are further classified into subclasses with respect to
the types of their H chains. The novel antibodies of
this invention embrace ones belonging to all these
classes and subclasses.
Moreover, the antibody of the invention does not
necessarily need to use the whole antibody molecule
and can use a portion of the molecule (active
fragment) insofar as it is active. As the active
fragment, concretely named are F(ab')2, Fab, Fv and
recombinant Fv. For example, F(ab')2 results if the
antibody is digested with pepsin, and Fab results if
digested with papain.
Although these fragments can be used alone, they
can be conjugated to substances such as albumin and
polyethylene glycol and can be used as new conjugates.
In many cases, such conjugates generally exhibit their
effectiveness to the maximum degree in vivo without
being decomposed for a long period of time. The
method for attaching a substance such as albumin or
polyethylene glycol to the active fragment is, for
example, described in "Antibodies A Laboratory
Manual," Cold Spring Harbor Laboratory, 1988, p. 77-81
and p. 129-137. In general, employing divalent-reactive
reagents such as SPDP (available from
Pharmacia Inc.), SMPB (available from Pierce Inc.),
and EMCS (Dotite Inc.), the active fragments can be
easily conjugated to albumin or the like.
Also, employing an active fragment, the primary
structure (e.g., Fc) other than the region (e.g.,
hypervariable region) necessary for reaction with the
TRAF family molecule in the H and L chains is
substituted by the corresponding primary structure of
an antibody derived from other animal species: this or
a similar method can yield chimera antibodies.
The novel antibodies of this invention can be
obtained according to methods known in the art,
regardless whether they may be polyclonal antibodies
or monoclonal antibodies. For example, they can be
obtained by referring to "Immunological Experimental
Procedures," edited and published by The Immunological
Society of Japan. With respect to the TRAF family
molecules of this invention to be used as immunogens,
the method of obtaining the TRAF family molecules
should not be questioned insofar as they have such
purity as will allow their use for preparation of the
antibodies.
Where the TRAF family molecule to be used as the
immunogen is a TRAF family molecule with a low
molecular weight or part thereof (namely a TRAF family
molecule comprising about 10 to about 20 amino acid
residues or a portion thereof), it may be conjugated
to a carrier such as keyholelymphethemocyanine (KLH)
and used as an antigen.
Animals to be immunized with the TRAF family
molecules or parts thereof may be any animal other
than human. Among animals that are used by those
skilled in the art, animal species capable of
producing the desired antibodies may preferably be
selected for use.
The polyclonal antibodies may be obtained by
purifying the resulting antisera. The purification
can be a combination of the methods such as salting
out, ion-exchange chromatography, and affinity
chromatography.
The monoclonal antibodies may be obtained by the
method for preparing hybridomas. For the cell fusion,
techniques using polyethylene glycol, Sendai virus,
electric pulse, etc. are available.
Other than the above, genetic engineering methods
can also be used to obtain the antibodies. For
example, mRNA is extracted from the spleen cells or
lymphocytes of an animal immunized with the TRAF
family molecule or part thereof, or from the hybridoma
producing a monoclonal antibody against the TRAF
family molecule or part thereof, from which a cDNA
library is prepared. The antibodies are expressed
using the cDNA library. The clones producing the
antibodies reactive to the antigens are obtained by
screening the cDNA library, the obtained clones are
cultured, and the desired antibodies can be purified
from the cultured mixture by the combination of
methods such as salting out, ion-exchange
chromatography, and affinity chromatography.
The antibodies of this invention can be used to
detect the TRAF family molecule or part thereof
existing in body fluids or tissues. The antibodies
can also be used to prepare antibody columns for use
in purification of the TRAF family molecule or part
thereof, as well as to detect the TRAF family molecule
or part thereof in their fractions.
(Recombinant Vectors and Transformants)
The recombinant vectors and transformants of this
invention are characterized by containing the whole or
parts of the genes encoding the TRAF family molecules.
These can be prepared according to a variety of
methods as shown in Examples 1, 4, 5, and 7. Any of
bacteria such as E. coli, yeast, animal cells can be
used as a host for the transformants.
Further, these may find their utility, for
example, in the production of the whole or part of the
TRAF5 molecule as shown in Examples 4 and 5, i.e., in
the method for production of a TRAF family molecule
according to this invention, or may be intended for
use in obtaining the whole or part of the TRAF5 gene
in a large quantity as shown in Examples 1 and 7.
(Method for Production of TRAF Family Molecule
According to This Invention)
According to the method of this invention for
production of a TRAF family molecule, the transformant
of the invention is first grown and the gene is
amplified and expressed. Next, the culture is
recovered and, if necessary, manipulations such as
concentration, solubilization, dialysis, and various
kinds of chromatography are conducted to purify the
TRAF family molecule of the invention.
A variety of references are available for the
cultivation of the transformants, and for example, it
can be carried out according to the method as
described in "Microorganism Experimental Methods," The
Japanese Society of Biochemistry, Incorporated
Foundation, ed. Tokyo Kagaku Dojin, 1992.
The purification methods involve salting out,
ultrafiltration, isoelectric precipitation, gel
filtration, electrophoresis, ion-exchange
chromatography, hydrophobic chromatography, a variety
of affinity chromatography such as antibody
chromatography, chromatofocusing, adsorption
chromatography, and reverse phase chromatography, and
may be carried out by selecting them as appropriate.
As the Examples illustrate in detail, the purification
is preferably done by means of affinity
chromatography using LT-βR and CD30 or that using the
antibodies of this invention.
Also, in the method for production, the TRAF
family molecule to be prepared may be produced by the
transformant as a fusion peptide with other
polypeptides. In this case, if necessary, the
manipulations of treatment with a chemical substance
such as bromocyanogen or an enzyme such as protease
and slicing out the TRAF family molecule are added to
the purification step.
(The antisense Polynucleotides of This Invention)
The antisense polynucleotides of this invention
embrace all of those in which a plurality of
nucleotides comprising bases, phosphoric acid, and
sugars are bonded, including those not present in
nature; their representatives are DNA and mRNA.
The antisense polynucleotides of the invention
find a use for the polynucleotide probes for research
purposes that aim at investigating the presence of
genes encoding the TRAF family molecules, as well as
the state of their expression.
Also, another use of the antisense
polynucleotides of the invention is to regulate the
expression of TRAF family molecules. It is expected
that the antisense polynucleotide hybridizes to DNA
encoding the TRAF family molecule or to mRNA encoding
the TRAF family molecule and promotes or suppresses
the expression of the TRAF family molecule. Therefore,
the antisense polynucleotides can be used as
therapeutic agents for disorders in the signal
transduction system involving the TRAF family
molecules or for disorders involving the LT-βR-mediated
signals. In other words, antisense drugs can
be developed based on the polynucleotides and
derivatives thereof.
Employing a polynucleotide containing the base
sequence complementary to DNA or mRNA that encodes a
protein, expression of the protein is to be regulated:
this method is referred to as "antisense method" and
is the technology currently being pursued by many
researchers. The polynucleotide having the
complementary sequence is believed to regulate
expression of the protein by binding to DNA or mRNA
that carries genetic information at any of the
following stages and influencing the normal flow of
transmission of genetic information: (1) transcription
stage from the gene to pre-mRNA; (2) processing stage
from the pre-mRNA to mature mRNA; (3) passing stage
through nuclear membranes; and (4) translation stage
to the protein.
In view of the ease of hybridization, it is
thought to be advantageous that polynucleotides or
polynucleotide derivatives having the base sequences
complementary to the base sequences of regions that
form stem loops be designed. (Clinical Immunology, vol.
25, p. 1200-1206, 1993.)
It is also expected that the polynucleotides
having the base sequences complementary to the base
sequences in the vicinity of translation start codons,
or at the sites of ribosomal binding, capping and
splicing have, in general, a great inhibitory effect
on expression. (Cancer and Chemotherapy, Vol. 20, No.
13, p. 1899-1907.) Thus, a great expression
regulatory effect is expected for the antisense
polynucleotides of this invention that contain the
genes encoding the TRAF family molecules or the base
sequences complementary to the base sequences of mRNA
against the genes in their vicinity of translation
start codons, or at their sites of ribosomal binding,
capping and splicing.
Also, for the antisense polynucleotide, the
length on the order of from 16 to 30 bases is
preferable for use in the regulation of expression.
(The polynucleotide Derivatives of This Invention)
The polynucleotides and antisense polynucleotides
of the invention embrace all those analogous to the
polynucleotides existing in nature with respect to
their stereochemical structures or functions, insofar
as the sense polynucleotides can express the TRAF
family molecules of the invention and the antisense
polynucleotides can hybridize to their complementary
polynucleotides (sense polynucleotides). For example,
they may be polynucleotides 3'- or 5'-termini of which
are bound to other substances, or may be substances
having undergone modifications such as substitution,
deletion, and addition in at least part of any of base,
sugar, and phosphoric acid of the polynucleotide, or
those having bases, sugars or phosphoric acid not
present in nature, or those having skeletons other
than sugar-phosphoric acid skeletons.
Also, the derivatives should preferably be ones
that have enhanced activity in at least one of
nuclease resistance, tissue selectivity, cell
permeability, and binding strength. It is known that
especially preferred polynucleotide derivatives have
phosphorothioate bonds as their skeleton structures.
The polynucleotides and derivatives thereof according
to this invention also embrace derivatives having
these functions or structures.
For example, among the derivatives, there are
ones which can be synthesized using a chemical
synthesizer (e.g., Type 394 available from Perkin
Elmer Inc.), such as the methylphosphonate type and
the phosphorothioate type. In this case,
manipulations are conducted following the manual as
attached to a chemical synthesizer and the synthesized
products as obtained are purified by HPLC methods such
as reverse phase chromatography; thus, the desired
polynucleotide derivatives can be obtained.
The contents of Application No. 034674/1996,
filed Feb. 22, 1996, in Japan is hereby incorporated
by reference.
EXAMPLES
This invention will be further detailed by
illustrating examples below; however, the invention is
not to be limited to these examples.
〈EXAMPLE 1〉 Identification of the TRAF5 Molecule
1. The Design of Primers
The base sequences for TRAF1 and TRAF2 were
screened for a high homology region. The portion from
Tyr to Gly of the amino acid sequence of TRAF1, namely
Tyr Leu Asn Gly Asp Gly (YLNGDG), was designated a
sense side and the amino acid sequence, Asp Asp
(Thr/Ala) (Ile/Met) Phe (Ile/Leu) (DD(T/A) (I/M) F
(I/L)), was designated an antisense side. With
respect to the sense side, a sense chain of the gene
encoding its amino acid sequence was synthesized as a
primer; with respect to the antisense side, an
antisense chain of the gene encoding its amino acid
sequence was synthesized as a primer. Here, "(I/M)"
as described in the above-mentioned amino acid
sequence means either of "I" and "M." The base
sequence encoding an amino acid sequence can not be
singularly determined because of degeneration: for
example, if the amino acid is Ala, there are four
conceivable cases-GCA, GCG, GCC, and GCT. In such a
case, the synthetic mode during the automated DNA
syntheses is selected to be GCX ("x" contains all of A,
G, C, and T) and a mixture of oligo-DNA primers that
correspond to the selected amino acid sequences was
prepared. These primers are shown in FIGs. 6A and 6B.
Here, FIG. 6A represents 256 kinds of the sense side
primer, whereas FIG. 6B represents 768 kinds of the
antisense side primer. The mixture of oligo- DNA
primers as described above contains all these.
Furthermore, each primer was designed to contain an
EcoRI site, as is shown in FIGs. 6A and 6B.
2. Preparation of cDNA Mixture
(1) Murine B cell lymphoma A20.25 (ATCC TIB208)
was grown in a 25-cm2 flask.
(2) After recovery and washing with PBS, the
cultured cells were suspended in 1 ml of PBS and the
cell number was counted.
(3) The cells (1x106) were taken into an aseptic
Eppendorf tube and centrifuged at 5,000 rpm for 3 min
to remove the supernatant. The pellet was subjected to
tapping.
(4) RNAZ01B (Registered Trademark, available from
CosmoBio Co. Ltd.) 200 µl was added and thoroughly
stirred with the tip of a pipetteman to dissolve the
cells.
(5) After 20 µl of chloroform was added and
shaken, the cells were allowed to stand in ice for 5
min. Subsequently, they were centrifuged at 15,000
rpm for 15 min at 4 °C to recover the colorless,
transparent portion of the upper layer, which was then
transferred to a new tube.
(6) Next, after centrifugation at 15,000 rpm for
15 min at 4 °C, the supernatant was discarded, 800 µl
of 75% ethanol was added to the pellet, and it was
allowed to stand at -20 °C for 30 min.
(7) Next, after centrifugation at 15,000 rpm for
15 min at 4 °C, 11.5 µl of distilled water was added
to the pellet.
(8) Next, oligo-dT (0.5 mg/ml) 0.5 µl was added
and it was allowed to stand at 70 °C for 10 min and on
ice for 5 min.
(9) Next, a solution having the composition as
described below was added and it was allowed to stand
at 42 °C for 50 min, at 90 °C for 5 min, and on ice
for 5 min.
5xRT Buffer | 4 µl |
10 mM dNTPmix | 1 µl |
Superscript RTase |
| 1 µl |
(Registered Trademark, available from GibcoBRL
Inc.) |
(10) RNaseH (Registered Trademark, available from
GibcoBRL Inc.) 1 µl was added and it was allowed to
stand at 37 °C for 20 min, affording the cDNA mixture.
3. PCR
(1) Employing the cDNA mixture obtained in 2 and
the oligo-DNA primers obtained in 1, PCR was performed
under the following conditions:
cDNA | 2 µl |
dNTPmix |
| 1 µl |
Primer (sense side)(100 pmol/1 µl) | 1 µl |
Primer (antisense side)(100 pmol/1 µl) | 1 µl |
10xPCR Buffer |
| 4 µl |
DDW | 30.5 µl |
Ampli-Taq | 0.5 µl |
Total |
| 40 µl |
Mineral oil (40 µl) was overlaid on the above
composition and it was allowed to stand at 94 °C for 5
min. Thereafter, the following cycle was repeated 40
times for reaction: at 45 °C for 2 min, followed by at
72 °C for 1 min, and followed by at 94 °C for 1 min.
Of note in this reaction was that the temperature of
annealing had been set at as low as 45 °C to design
such that low homology ones could be picked up as many
as possible.
(2) After reaction, the PCR product was
electrophoresed on a mini gel (1.5% agarose gel).
(3) A band was sliced out from the gel: the band
was believed to be the gene encoding the about 0.5 kb
TRAF domain (the portion of from position 342 to
position 558 of the amino acid sequence of SEQ ID No.
1 of the Sequence Listing). PCR product was recovered
from this by means of Gene CleanII (Registered
Trademark, available form BIO 101 Inc.).
(4) Further, part of the recovered product was
again subjected to the mini gel electrophoresis as
described above, thus ascertaining that a band
appeared at about 0.65 kb.
4. DNA Ligation
Employing a Bluescript vector (available from
Stratagene Inc.), DNA ligation was carried out in a
reaction medium having the following composition:
ADDW | 5 µl |
10xLigation buffer |
| 1 µl |
PCR Vector |
| 2 µl |
PCR Product |
| 1 µl |
T4DNA Ligase |
| 1 µl |
Total | 10 µl |
Reaction was carried out at 14 °C overnight to
give a ligation mixture.
5. Transformation
Transformation was performed using a Bluescript
cloning kit.
(1) To a cell suspension (5 µl) on ice were added
2 µl of 0.5 M β-mercaptoethanol and the ligation
mixture prepared in (4), and allowed to stand for 30
min. Subsequently, it was allowed to stand in a hot
water bath at 42 °C for 30 sec and then, on ice for 20
min. A SOC medium (450 µl) was added, and it was
incubated at 225 rpm for 1 h at 37 °C. (2) Next, the incubated medium was spread onto LB
agar plates (+ampicilline, X-Gal, and IPTG) at 50 µl,
100 µl, and 200 µl, respectively. (3) After incubation at 37 °C for 18 h, the
incubated products were allowed to stand at 4 °C for 2
h and colonies in white and in blue appeared.
6. Culturing on Miniscale
(1) Ninety-six white colonies were picked up from
the respective plates prepared in 5.
(2) One of the aforementioned colonies was added
to a 3-ml LB medium (+Amp) and shaken at 37 °C
overnight.
7. Preparation on Miniscale
According to the method as described in
"Molecular Cloning 2nd Edition," Cold Spring Harbor
Laboratory, 1989, p. 1.25-1.28, the following
manipulations were conducted.
(1) The cultured preparation (1.5 ml) was taken
into an Eppendorf tube: the remaining preparation was
spread, and cultured at 37 °C and preserved. It was
centrifuged at 6,000 rpm for 2 min at 4 °C. (2) Solution 1, 100 µl (lysozyme 5 mg/ml) was
added to the pellet and it was allowed to stand at
room temperature for 5 min. Subsequently, Solution 2
(200 µl) that had been gently mixed on ice for 5 min
was added, Solution 3 (150 µl) that had been mixed on
ice for 15 min was added and next, centrifugation was
carried out at 12,000 rpm for 5 min at 4 °C. For the
composition of each solution, the above-mentioned
"Molecular Cloning 2nd Edition" was followed. (3) The supernatant was taken into a new
Eppendorf tube. To this was added an equal volume of
phenol, and then, centrifugation was carried out at
12,000 rpm for 1 min at room temperature. (4) The supernatant was taken into a new
Eppendorf tube. To this was added an equal volume of
a mixture of CHCl3: iAA (99:1) and then, centrifugation
was carried out at 12,000 rpm for 1 min at room
temperature. (5) The supernatant was taken into a new
Eppendorf tube. To this was added 1 µl of Mussel
glycogen and 900 µl of ethanol and after allowing it
to stand at -80 °C for 30 min, centrifugation was
carried out at 15,000 rpm for 5 min at 4 °C. (6) The precipitates were dried. To this were
added 20 µl of TE and 1 µl of RNase A (5 mg/ml) and it
was allowed to stand at 65 °C for 20 min, affording a
plasmid DNA. (7) Electrophoresis on a minigel was conducted
under the conditions as described below to examine the
bands.
H Buffer | 1 µl |
EcoRI |
| 1 µl |
DNA |
| 1 µl |
ADDW |
| 7 µl |
Total | 10 µl |
After the plasmid DNA was incubated in the above-mentioned
solution at 37 °C for 1 h, it was added onto
a 0.75% agarose gel and electrophoresed.
8. DNA Sequencing
(1) Plasmid DNA (1 µl) was taken up and diluted
with 99 µl of TE.
(2) The absorbance (A260) was measured with an
spectrophotometer at 260 nm and DNA values were
determined: the DNA concentration (DNA value) of a
diluted solution when A260 is 1.0 is 50 µg/ml.
(3) Based on the A260 values, the DNA was diluted
with TE so that it reached 1 µg/µl.
(4) DNA sequencing was conducted using a DNA
Sequencer Model 373A (available from ABI) according to
the dyeterminator method.
(5) Following the above-mentioned method,
homology was investigated with respect to the 96
clones screened in 6, and as a result, 10 clones (all
having the same sequence) of a novel gene belonging to
the TRAF family were obtained.
As noted above, part of the novel gene belonging
to the TRAF family was provided by lowering the
annealing temperature during PCR to 45 °C.
9. Construction of cDNA Library
Employing a Uni-ZAP (Registered Trademark,
Stratagene Inc.) a cDNA Library kit (available from
Stratagene Inc.) and a Poly(A) quick mRNA isolation
kit (available from Stratagene Inc.), a cDNA library
was constructed according to their respective manuals.
(1) Murine monocyte cell strain J774A.1 (ATCC
TIB-67) 1x108 cells were made into total RNA by means
of the Uni-ZAP kit. (2) Using this RNA and the Poly(A) quick mRNA
isolation kit (available from Strategene Inc.), 10 µg
of Poly(A)RNA was finally obtained. (3) From this Poly(A)RNA, the cDNA library was
constructed using the Uni-Zap kit as previously
described.
Other than the present examples, preparation of
the Poly(A)RNA and cDNA libraries was also feasible by
consulting the protocols as described in references
listed in the following: "Molecular Cloning 2nd
Edition," Chapter 8; and "Preparation of Gene
Libraries; Biomanual Series 2, p. 33-40, and 95-106,
Yodo Co. Ltd.
10. Screening for cDNA
In order to obtain cDNA corresponding to the PCR
product (clones) that was obtained through Steps 1-8
and that was considered belonging to the TRAF family,
hybridization between the PCR product and the cDNA
library constructed in 9 was carried out, and a novel
cDNA of the novel TRAF family was screened for in the
manner described below. The hybridization conditions
adopted those as described in "Cell Engineering
Experimental Protocols," p. 57-65, Shujun Co. Ltd.,
1991.
(1) Employing a random primer labeling kit
(available from Takara Shuzo Co. Ltd.) and a [γ-32P]ATP
(available from Amasham Inc.), the PCR product
obtained in 7 was labeled with 32P, which served as a
DNA probe. (2) Next, the cDNA obtained in 9 was screened
according to the plaque hybridization method (see,
"Cell Engineering Experimental Protocols," p. 57-65,
Shujun Co. Ltd., 1991.), affording 16 kinds of cDNA
clones. (3) DNA sequencing was conducted on these 16
kinds of clones, and finally, one clone was selected
by excluding clones with partial lengths and those
with overlapping sequences. The base sequence of this
clone is set forth in SEQ ID No. 2 of the Sequence
Listing. Transformed E. coil into which this cDNA was
introduced was deposited on February 9, 1996 in the
National Institute of Bioscience and Human-Technology,
Agency of Industrial Science and Technology, Ministry
of International Trade and Industry locating at 1-1-3
Higashi, Tsukuba, Ibaragi, 305 Japan (Accession No:
FERM BP-5384). (4) A computer analysis was performed on the
basis of the base sequence of this clone with the full
2224 bp length. As a result, the cDNA was found to
contain an open reading frame with 1674 bp. In other
words, it was found that the protein encoded by the
cDNA comprised a sequence of 558 amino acids. The
amino acid sequence is set forth in SEQ ID No. 1 of
the Sequence Listing. (5) Also, as a result of homology comparison
between the protein and other TRAF family molecules,
it was determined that the protein had homology of 29-42%
in its overall length, as is shown in FIG. 1.
Particularly, the TRAF domain (the portion of from
position 342 to position 558 of the amino acid
sequence set forth in SEQ ID No. 1 of the Sequence
Listing) had homology of 40-55%. From these results
and the protein structure shown in FIG. 1, it was
ascertained that the protein is evidently a novel
molecule belonging to the TRAF family molecule. Thus,
the protein was identified as a novel TRAF family
molecule and designated TRAF5 molecule. In FIG. 1,
the blacked part represents a TRAF domain (the portion
of from position 342 to position 558 of the amino acid
sequence of TRAF5), the vertically lined part
represents a leucine zipper motif (the portion of from
position 261 to position 303 of the amino acid
sequence of the TRAF5 molecule), the obliquely lined
part represents a Zn finger motif (the portion of from
position 111 to position 250 of the amino acid
sequence of the TRAF5 molecule), and the horizontally
lined part represents a Zn ring finger motif (the
portion of from position 45 to position 85 of the
amino acid sequence of TRAF5).
〈EXAMPLE 2〉 Investigation of Expression of the TRAF5
Molecule in Tissues
As to which tissue TRAF5 was expressed in was
investigated in the following manner:
(1) Tissues were extracted from murine brain,
thymus, lung, liver, spleen, heart, stomach, and
adrenal gland, and then, respective total RNAs were
extracted with RNAZ01B. (2) Subsequently, employing a Poly(A) quick mRNA
isolation kit (available from Stratagene Inc.),
Poly(A) RNAs were prepared according to the same
protocol as 9 in Example 1. (3) For these Poly(A) RNAs, the RNA was denatured
by heating it in 50% formamide at 65 °C for 5 min,
electrophoresed on a 1.5% agarose gel containing 6.6%
formamide and transferred to a nitrocellulose membrane
(also, a nylon membrane usable). (4) The DNA probe prepared in 10.1 of Example 1
was used as a 32P-probe. Hybridization manipulations
were carried out under the high stringent conditions
according to "Molecular Cloning A Laboratory Manual
2nd Edition," p. 7.39-7.52. The results are shown in
FIG. 2. "28S" indicates the position of 4.9 kb and
"18S" indicates the position of 1.9 kb. mRNA bands
were observed at the positions of 2.2 kb (as indicated
by the arrow) in all the tissues, thus ascertaining
expression of the TRAF5 gene.
〈EXAMPLE 3〉 Screening for TRAF5-Associated Substances
Screening was conducted for any substances among
the TNF-R family molecules that associate TRAF5.
(1) As regards TNF-R1, TNF-R2, CD40, and LT-βR
among the TNF-R family molecules, a fusion protein
with GST was prepared by inserting into the downstream
region of glutathione-S-trasnferase (GST), a domain
part within the cytoplasm of each TNF-R family
molecule. (2) Employing an In vitro transcription and
translation system (TNT available from Promega Inc.),
TRAF5 was translated in vitro following its attached
manual. Electrophoresis ascertained that the TRAF 5
in vitro translated product was labeled with 35S-methionine. (3) Next, the TRAF5 in vitro translated product
was mixed with GST and each kind of the fusion
proteins prepared in (1). (4) Subsequently, the mixture was added to a GSH
agarose column, and GST and the GST-TNF-R family
molecule (which may contain any associated substance
with the TRAF5 molecule) were allowed to adsorb to the
column. (5) Then, beads in the GSH agarose column were
added to a SDS sample buffer and boiled at 95 °C for 5
min in order to dissociate the TRAF5 molecule that was
associating any GST-TNF-R family molecule therefrom. (6) Then, after the beads were precipitated by
centrifugation, the supernatant was subjected to SDS-PAGE.
A 10% gel was used as a gel, and after
electrophoresis, it was incubated in an Amplify
(Registered Trademark, available from Amasham Inc.)
for 30 min. (7) Thereafter, the gel was dried and exposed to
an X-ray film. The results are shown in FIG. 3. The
"In vitro pro" lane represents a lane for the in_vitro
translated product in FIG. 3. In addition, the name
of each kind of the fusion proteins, which was used,
is entered in each lane. As is evident from FIG. 3, a
band corresponding to the TRAF5 molecule at about 50
kD could be detected in the case of reaction with the
GST-fusion protein of LT-βR. In other words, the
TRAF5 molecule was found to associate LT-βR.
〈EXAMPLE 4〉 Immunoprecipitation Western Blotting
According to the immunoprecipitation western
blotting, association between the TRAF5 molecule and
LT-βR was ascertained.
(1) First, the hemagglutinin epitope (HA epitope)
of influenza virus was bound to the TRAF5 molecule as
a tag in the following manner:
(i) An oligonucleotide corresponding to nine
amino acids of the HA epitope was synthesized.
Specifically, formula (1) as described below was
synthesized as a sense polynucleotide and formula (2)
as an antisense polynucleotide by means of a DNA
synthesizer.
Formula (1) 5'-ATT TGA TGT ACC CAT AGG ATG TTC CAG ATT
ACG CTG ATT TCC-3' Formula (2) 5'-TCG AGG AAT TCA GCG TAA TCT GGA ACA TCG
TAT GGG TAC ATC-3' (ii) These polynucleotides were mixed and
annealed at 50 °C for 10 min. (iii) Next, phosphorylation was carried out using
a T4 polynucleotide kinase (available from Promega
Inc.). (iv) This was subcloned into a vector plasmid
pMKITneo that had been treated with restriction
enzymes-EcoRI and XhoI, the resulting clone was
designated pHAKITneo. (v) The pHAKITneo was treated with EcoRI and the
TRAF5 gene that had been amplified by PCR was
subcloned into it. As compared to direct introduction
of the TRAF5 gene into pMKITneo by EcoRI treatment,
reading can be well done in inframe when TRAF5 is
introduced into pHAKITneo. (2) Next, 2.5 µg of the pHAKITneo-TRAF5 gene and
pcDNA1-LT-βR (LT-βR-introduced vector) were
transiently introduced into COS7 Cell (ATCC CRL-1651)
2x106 cells, respectively by the DEAE-dextran method
(available from Pharmacia Inc.). (3) After transformants of the COS7 cells were
grown for 48 h and the proteins encoded by the
introduced genes were allowed to produce, the cells
were recovered and dissolved with a cytolytic solution.
For the cytolytic solution, a solution containing 0.1%
NP-40, 50 mM HEPES (pH 7.4), 250 mM NaCl, 10%
glycerol, 2 mM EDTA, 1 mM PMSF, 2 µg/ml aprotinin, 2 µg/ml
Pepstatin A, and 2 µg/ml leupeptin was used. (4) The anti-LT-βR antibody was allowed to act on
a solution into which these cells had been dissolved
and the TRAF5-LT-βR complex was immunoprecipitated.
(i) Preclearing was conducted against the
supernatant (1 ml) of the cell-dissolved solution with
the aid of Sepharose beads bound to goat IgG. Namely,
100 µl of goat Sepharose beads (equivalent to 50 µl
of beads) was added to 1 ml of the supernatant and
they were allowed to react at 4 °C for 16 h.
Thereafter, the beads were removed by centrifugation
at 6,000 rpm for 2 min and any substances bound to the
goat IgG in nonspecific manner were thus removed. (ii) Next, 100 µg of Sepharose beads (equivalent
to 50 µl of beads) to which the anti-LT-βR antibody
was bound was added to the supernatant and they were
allowed to react at 4 °C for 16 h. Thereafter, parts
that did not adsorb to the beads were removed by
centrifugation, and further, these beads were
centrifuged in the cytolytic solution used in (3)
twice. (5) These beads were put into 20 µl of a reductive
sample buffer for SDS-PAGE and boiled for 5 min. (6) Subsequently, electrophoresis was conducted
using a concentration-gradient gel for SDS-PAGE
(acrylamide gel concentration: 10-20%). (7) After electrophoresis, the gel was
transferred onto a nitrocellulose membrane at 10 V at
4 °C overnight. (8) To this membrane was added Blockace
(available from Dainippon Pharmaceutical Co. Ltd.) and
blocking was conducted at room temperature for 1 h. (9) Next, after the membrane was washed with PBS
(pH 7.4) containing 0.05% Tween20 for 10 min, the goat
anti-HA antibody (available from Boehringer Inc.) was
added at 10 µg/ml and it was allowed to react at room
temperature for 1 h. It was then washed with Tween
PBS for 10 min. These manipulations were repeated
three times. (10) Next, a solution containing peroxidase-labeled
goat IgG (available from Caltag Inc.) diluted
1000-fold was added to the membrane and it was allowed
to react at room temperature for 1 h. After the
reaction, the membrane was washed with Tween PBS three
times, and the coloring solution from an ECL kit
(available from Amasham Inc.) was added and it was
allowed to react for 1 min. (11) This membrane was exposed to an X-ray film
for 20 sec and developed. The result is shown in FIG.
4. "N.G.S." represents a lane for the goat serum and
"Anti-LT-βR" represents a lane for the sample obtained
by treatment of the immunoprecipitated product with a
reductive sample buffer for SDS-PAGE. As is evident
from FIG. 4, the band of the HA-TRAF5 molecule at
about 60 kD (as indicated by the arrow) can be
recognized for the product immunoprecipitated
with the anti-LT-βR antibody.
〈EXAMPLE 5〉 Characterization of the TRAF5 Molecule
(investigation of activation for the DNA-binding of
NF-κB)
As to what kind of signals the TRAF5 molecule
transmits within the nuclei of cells, investigation
was made by studying activation of the DNA-binding of
NF-κB.
(1) The three vectors as described below (7.5 µg
each) were introduced into 293 Cell 2x106 cells,
respectively so that the five expression patterns as
described below could be realized. The introduction
employed CaPO4 and the TRAF5 molecule was transiently
expressed in the cells.
Vectors Employed:
(i) Vector obtained by insertion of the full-length
cDNA of the TRAF5 gene into a pMKITneo vector;
(ii) Vector obtained by insertion of the fragment
of from position 233 to position 558 of the TRAF5 gene
into a pMKITneo; and
(iii) Vector obtained by insertion of LT-βR into
a pMKITneo vector.
Expression Patterns:
(i) pMKITneo Vector only;
(ii) pMKITneo Vector-the full length TRAF5 gene;
(iii) pMKITneo Vector-positions 233-558 of the
TRAF5 gene;
(iv) pMKITneo Vector-LT-βR; and
(v) pMKITneo Vector-positions 233-558 of the
TRAF5 gene and pMKITneo Vector-LT-βR (the ratio of the
former to the latter: 10:1).
- (2) Forty-eight hours after the introduction,
each 4 µg of a nuclear extract was obtained. (See,
"Biomanual Series 5 Methods for Studying Transcription
Factors" p. 17-26, Yodo Co. Ltd., 1993.)
- (3) Polynucleotides of formulae (3) and (4) as
described below, which are the binding sites of NF-κB,
were respectively synthesized, and these were made
into a double-strand, 5'-end of which was labeled with
32P by means of Megarabel (Registered Trademark,
available from Takara Shuzo Co. Ltd.) to prepare a DNA
probe for the detection of NF-κB.
- Formula (3) 5' ATCAGGGACTTTCCGCTGGGGACTT 3'
- Formula (4) 5' CGGAAAGTCCCCAGCGGAAAAGTCCC 3'
(4) The extract of (2) and the probe prepared in
(3) were reacted at 37 °C for 30 min.
(5) After the reaction, electrophoresis was
performed in an Electrophoretic Mobility Shift Assay
(EMSA) to study the DNA-binding ability of NF-κB. The
results are shown in FIG. 5. Lanes 1-5 correspond to
the expression patterns (i)-(v) as described above.
The band as indicated by "B→" represents the NF-κB
bound to the DNA probe for detection. As is evident
from FIG. 5, NF-κB is detected by having been bound to
the DNA probe if the TRAF5 molecule is expressed.
From this, it can be understood that the expression of
the TRAF5 molecule induces activation of the DNA-bonding
of NF-κB. Although the expression of LT-βR
alone also induces the expression of NF-κB, it has
been found that if the portion of from position 233 to
position 558 of the amino acid sequence of the TRAF5
molecule, which is a deleted protein, is added in an
about 10 times excess, this activation can be
inhibited. In other words, the DNA-binding activity
of NF-κB can be inhibited by the excessive addition of
the portion of from position 233 to position 558 of
the amino acid sequence of the TRAF5 molecule. This
suggests that preparation of the TRAF5 molecule can
control the signal transduction.
〈EXAMPLE 6〉 Preparation of Antibodies
1. Preparation of Antigens
The two peptides as described below, which are
parts of the TRAF5 molecule, were synthesized using a
peptide synthesizer.
Sequence 1: 14mer peptide comprising Ser Val Lsy Gln
Arg Ile Thr Gln Leu Glu Ala Ser Asp (SVKQRITQLEASD) of
from position 351 to position 363 of the TRAF5
molecule the N-terminus of which is appended to Cys
(C) Sequence 2: 14mer peptide comprising Cys Tyr Ser Gly
Lys Leu Ile Trp Lys Val Thr Asp Tyr Arg
(CYSGKLIWKVTDYR) of from position 401 to position 414
of the TRAF5 molecule
2. Preparation of Polyclonal Antibodies
The antibodies were prepared independently
against the respective peptides prepared in 1 by
following the procedure as described below.
(1) The synthesized peptide (5 mg) was conjugated
to 10 mg of KLH (available from Wako Pure Chemicals co.
Ltd.) by means of m-maleimidobenzoyl-N-hydroxysuccinimide
ester (available from Pierce Inc.)
and it was mixed with Freund complete adjuvant
(available from Difco Inc.) in the ratio of 1:1 to
provide an emulsion. (2) Rabbits were immunized by intramuscularly
injecting the above-mentioned emulsion in an amount of
its one sixth per animal. (3) The same operation was repeated twice every
two weeks. Where a polyclonal antibody against a
rabbit was to be prepared, blood was collected seven
days after the final immunization and serum was
separated. (4) The serum was further salted out with 40%
saturated ammonium sulfate and an IgG fraction
(polyclonal antibody) was obtained by affinity
chromatography using Protein A Sepharose (available
from Pharmacia Inc.). The serum (50 ml) produced 270
mg of the polyclonal antibody. (5) Measurement of the titers for the polyclonal
antibodies was carried out by ELISA following the
method as described below.
- (i) Each of the antigen preparations (peptide of
sequence 1 and peptide of sequence 2) 25 µg/ml was
fractionally poured to each well of 96-well ELISA
plate (Xenobind, available from Xenopore Inc.) at 50 µl
per well, and they were allowed to stand at 4 °C
overnight.
- (ii) The antigen preparation was discarded and
Blockace (available from Dainippon Pharmaceuticals Co.
Ltd.) diluted 4-fold with distilled water was
fractionally poured to each well at 200 µl per well,
blocking was performed and it was allowed to stand at
room temperature for 2 h.
- (iii) The blocking solution was discarded. The
polyclonal antibodies diluted 160-, 320-, 640-, 1280-,
2560-, 5120-, and 10240-fold and the serum from a
normal nonimmunized rabbit were fractionally poured to
eight wells, respectively at 100 µl per well, and they
were allowed to stand at room temperature for 1 h.
- (iv) The culture supernatant was discarded and
the plate was washed with 0.05% Tween20-PBS four times.
The biotinylated anti-rabbit IgG antibody (available
from Vector Inc.) was fractionally poured to each well
at 50 µl per well, and it was allowed to react at room
temperature for 1 h.
- (v) The plate was washed with 0.05% Tween20-PBS
four times. An ABC solution (avidin-biotinylated
peroxidase mixture, available from Vector Inc.) was
fractionally poured to each well at 50 µl per well,
and it was allowed to react for 30 min.
- (vi) The plate was washed with 0.05% Tween20-PBS
four times. H2O2-OPD/PCB was fractionally poured to
each well at 100 µl per well, and coloring was allowed
to occur: A490 values were measured.
These results are shown in FIGs. 7 and 8. FIG. 7
is a graph showing the results of an ELISA for the
polyclonal antibody against the peptide of sequence 1.
FIG. 8 is a graph showing the results of an ELISA for
the polyclonal antibody against the peptide of
sequence 2. In the figures, the axis of abscissas
represents concentrations of the added polyclonal
antibody and the axis of ordinates represents A490
values. "Peptide of Sequence 1" represents the
results of an ELISA for the polyclonal antibody
obtained by immunization of two rabbits. From the
fact that A490 values are raised by addition of the
antibody, it can be understood that the antibody has
reacted with the antigen.
〈EXAMPLE 7〉 Cloning of the Human TRAF5 Gene
1. Preparation of cDNA Library
The preparation of a cDNA library was carried out
by a method similar to 9 of Example 1. However,
HAT109 Cell into which the gene of human HLTV-1 had
been introduced (provided by Dr. Yoshida at Medical
Sciences Research Institute of the University of
Tokyo) and a λgt11 cDNA Library kit (available from
Stratagene Inc.) were employed, respectively instead
of murine mocyte cell strain J777A.1 cells and the
Uni-ZAP cDNA Library kit (Registered Trademark,
available from Stratagene Inc.).
2. Screening for cDNA
cDNA for the portion of from position 1646 to
position 1966 of the base sequence set forth in SEQ ID
No. 2 of the Sequence Listing was labeled with [α
32P]dCTP using a Rediprime kit (available from Amasham
Inc.). The cDNA library prepared in 1 was screened
according to the plaque hybridization method (See,
"Cell Experimental Protocols," p. 57-65), using this
cDNA as a probe. Nineteen positive clones resulted.
DNA sequencing was conducted on all of these clones
and the base sequence of each clone was determined.
This result revealed that the 19 clones were the
fragments of one full-length gene. From these
fragments, the base sequence of the human TRAF5 gene
was determined. The base sequence is set forth in SEQ
ID No. 4 of the Sequence Listing. The amino acid
sequence of human TRAF5 is also set forth in SEQ ID No.
3 of the Sequence Listing.
Transformed E. coli into which the longest gene
(which contains positions 421-1860 of SEQ ID No. 4 of
the Sequence Listing) out of the 19 clones had been
introduced was deposited on February 14, 1996 in the
National Institute of Bioscience and Human-Technology,
Agency of Industrial Science and Technology, Ministry
of International Trade and Industry locating at 1-1-3
Higashi, Tsukuba, Ibaragi, 305 Japan (Accession No:
FERM BP-5821). Further, concerning the portions that
do not reached the full length, information was
obtained from other clones.
When human TRAF5 is compared to the structure of
murine TRAF5 molecule with regard to the positions of
respective domains, it is recognized that the TRAF
domain is positions 342-557, the leucine zipper motif
is positions 261-303, the Zn finger motif is positions
111-250, and the ring finger motif is positions 45-85.
〈EXAMPLE 8〉 Cross-Reaction of Antibodies
The reactivity of the anti-murine TRAF5 antibody
prepared in Example 6 was investigated.
1. Dissolution of Cells
First, for BJAB (Burkitt's lymphoma), HEK293
(cell strain derived from human fetus), ME180
(cervical carcinoma) and FDC-1 (follicular dendritic
cell), the cells were dissolved after their recovery
in the following manner. With respect to each, 1x106
cells were used. The cytolytic preparation contains
50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 50 mM
iodoacetamide, 2 mM MgCl2, 2 mM CaCl2, 0.1% NaN3, 1 mM
EGTA, 1 mM EDTA, 1 mM Na3VO4, and 1 mM NaF. This
cytolytic preparation (100 µl) was added to pellets of
the respective cells and stirred thoroughly to
dissolve the cells.
After allowing to stand on ice for 60 min,
centrifugation was conducted at 15,000 rpm for 10 min
to recover the supernatant.
2. Removal of Nonspecific Binding Proteins
To the recovered supernatant was added 50 µl of
beads in which IgG (1 mg) of a nonimmunized rabbit
was bound to CNBr-activated Sepharose beads (available
from Pharmacia Inc.). After reaction at 4 °C
overnight, centrifugation of the supernatant was
conducted at 15,000 rpm for 5 min to remove
nonspecific binding proteins.
3. Electrophoresis
The solution obtained in 2, 10 µl, was mixed with
an equal amount of a SDS-PAGE sample buffer and 1 µl
of 2-mercaptoethanol, heat treatment at 95 °C for 5
min was carried out, and then, it was electrophoresed
on a 5-20% gradient gel.
4. Membrane Transfer
After electrophoresis, the gel was transferred
onto a nitrocellulose membrane with the aid of a Trans
Blot System (Marisol Inc.).
5. Blocking
The nitrocellulose membrane was immersed in
Blockace (available from Dainippon Pharmaceuticals
Inc.) and allowed to stand for 1 h to effect blocking.
Thereafter, it was twice washed with 0.05% Tween20/PBS
for 5 min.
6. Antigen-Antibody Reaction
After the anti-murine TRAF5 antibody was diluted
with PBS to 1 µl/ml, it was added to the membrane and
allowed to react at room temperature for 2 h.
Thereafter, it was three times washed with 0.05%
Tween20/PBS for 5 min.
7. Coloring Reaction
Next, a solution containing peroxidase-labeled
anti-rabbit IgG (available from Caltag Inc.) that was
diluted with PBS 1000-fold was added to the
nitrocellulose membrane and it was allowed to react at
room temperature for 1 h. Thereafter, the membrane
was washed three times with 0.05% Tween20/PBS for 5
min.
Next, 5 ml of the coloring solution from an ECL
kit (available from Amasham Inc.) was added to the
membrane and it was further allowed to react for 1 min.
Subsequently, this nitrocellulose membrane was exposed
to an X-ray film for 20 sec, the film was developed,
and then, a photograph was taken. The result is shown
in FIG. 9. In FIG. 9, the band of the TRAF5 molecule
is observed at the position of about 65 kD in FDC-1
cells. From this, it has become apparent that the
anti-murine TRAF5 antibody obtained in Example 4 also
cross-reacts the human TRAF5 molecule.
〈EXAMPLE 9〉 Screening (2) for TRAF-Associated
Substances
Similarly to the method of Example 3, association
between TRAF5 and CD30 was investigated. Also, with
respect to TRAF2 and TRAF3, their association with
CD30 and LT-βR was investigated in like manner. The
results are shown in FIG. 10. As is evident from the
figure, it is understood that TRAF5 associates CD30
and that both of TRAF2 and TRAF3 associate either of
CD30 and LT-βR. In addition, in FIG. 10 "mock"
represents the cell into which a vector plasmid having
no insertion was introduced.
Concerning TRAF1, TRAF2, TRAF3, TRAF4, and TRAF5,
Table 1 summarizes their functions and structural
characteristics obtainable from the combination of the
already-known findings and the results from Examples 3
and 5. In the table, "+" means "having the
characteristics," "-" means "not having the
characteristics," and "N.D." means "can not be
determined either way."
| TRAF1 | TRAF2 | TRAF3 | TRAF4 | TRAF5 |
association with TNFR-2 | + | N.D. | + | N.D. | - |
association with CD40 | + | N.D. | + | N.D. | - |
association with LT-βR | N.D. | + | + | N.D. | + |
association with CD30 | N.D. | + | + | N.D. | + |
the ability to induce activation of NF-κB | - | + | - | - | + |
having leucine zipper motif | - | - | + | - | + |
having coiled coil structure | - | - | + | - | + |
INDUSTRIAL APPLICABILITY
The TRAF family molecules of this invention are
useful to elucidate the functions of the TNF-R family
molecules. The TNF-R family molecules and TNF are a
group of important molecules that are the trigger for
causing differentiation, proliferation and necrosis
within the cells. Thus, if the TRAF family molecules
are used to elucidate the foregoing phenomena, it will
presumably lead to elucidation of the mechanisms for
differentiation of the immune system, cancer and
apotosis.
The TRAF family molecules of this invention are
also useful for elucidation of the signal transduction
from LT-βR and as immunomodulators for the signals of
LT-βR.
The antibodies of this invention enable the
detection of the TRAF family molecules existing in
body fluids and tissues. The antibodies also enable
the preparation of antibody columns useful to purify
the TRAF family molecules of this invention, as well
as enable the detection of the TRAF family molecules
in respective fractions.
Active fragments of the antibody of this
invention enable the preparation of chimera antibodies.
Polynucleotide probes for research and diagnosis
that aim at investigating the presence of the genes
encoding the TRAF family molecules and their
expression state in tissues and cells are provided by
the genes encoding the TRAF family molecules of this
invention, the polynucleotides comprising the base
sequences of the antisense chains of the genes, parts
thereof and derivatives thereof.
It is also possible to regulate the expression of
TRAF family molecules by the genes encoding TRAF
family molecules of this invention, the polynucleotide
comprising the base sequences of the antisense chains
of the gene, parts thereof and derivatives thereof.
In other words, the TRAF family molecules and
derivatives thereof provide therapeutic agents for
disorders of the signal tranduction system involving
the TRAF family molecules, as well as for the
disorders involving the LT-βR- and CD30-mediated
signals. That is, it is possible to develop antisense
drugs based on the polynucleotides and derivatives
thereof.